Accumulation of Armadillo Induced by Wingless, Dishevelled, and Dominant-negative Zeste-white 3 Leads to Elevated DE-cadherin inDrosophila Clone 8 Wing Disc Cells

Abstract

Drosophila genetic studies suggest that in the Wingless (Wg) signaling pathway, the segment polarity gene products, Dishevelled (Dsh), Zeste-white 3 (ZW-3), and Armadillo (Arm), work sequentially; wg and dsh negatively regulate zw-3, which in turn down-regulates arm. To biochemically analyze interactions between the Wg pathway and Drosophila E-cadherin (DE-cadherin) which bind to Arm, we overexpressed Dsh, ZW-3, and Arm, in the Drosophila wing disc cell line, clone 8, which responds to Wg signal. Dsh overexpression led to accumulation of Arm primarily in the cytosol and elevation of DE-cadherin at cell junctions. Overexpression of wildtype and dominant-negative forms of ZW-3 decreased and increased Arm levels, respectively, indicating that modulation in zw-3 activity negatively regulates Arm levels. Overexpression of an Arm mutant with an aminoterminal deletion elevated DE-cadherin levels, suggesting that Dsh-induced DE-cadherin elevation is caused by the Arm accumulation induced by Dsh. Moreover, the Dsh-, dominant-negative ZW-3-, and truncated Arm-induced accumulation of DE-cadherin protein was accompanied by a marked increase in the steady-state levels of DE-cadherin mRNA, suggesting that transcription of DE-cadherin is activated by Wg signaling. In addition, overexpression of DE-cadherin elevated Arm levels by stabilizing Arm at cell-cell junctions.

Wingless signaling modulates cadherin-mediated cell adhesion inDrosophilaimaginal disc cells

“…We found that the endogenous DE-cadherin gene was transcriptionally activated in cl8Dshmyc cells only after prolonged overexpression of Dsh. Similar observations had been reported earlier (Yanagawa et al, 1997). Interestingly, in mouse hair follicles, Wnt signaling changes E-cadherin gene expression in the opposite manner, as it lowers E-cadherin transcription (Jamora et al, 2003).…”

Identification and Characterization of a Novel Line of Drosophila Schneider S2 Cells That Respond to Wingless Signaling

“…Immunoblot Analysis and Antibodies-Clone 8, S2Rϩ, and S2 cells grown to 80% confluence were lysed in lysis buffer and used to prepare samples for SDS-polyacrylamide gel electrophoresis, as described (22). The samples were loaded on 7.2% SDS-polyacrylamide gels, and the gels were then Western-blotted (21).…”

“…The samples were loaded on 7.2% SDS-polyacrylamide gels, and the gels were then Western-blotted (21). The antibodies used for Western analysis included the mouse monoclonal anti-Arm antibody N2-7A1 (29), the rat polyclonal anti-Dsh region I antibody (21), affinity purified rabbit anti-Wg IgG (30), the rat monoclonal anti-DE-cadherin antibody DCAD-2 (31), the rat monoclonal anti-Drosophila-␣-catenin antibody DCAT-1 (32), and peroxidase-conjugated secondary antibodies against mouse IgG (Bio-Rad), rat IgG (Jackson ImmunoResearch, West Grove, PA) and rabbit IgG (Cappel, Durham, NC), and the antibodies were diluted as described (22). Blots were visualized with enhanced chemiluminescence reagent (ECL, Amersham, UK).…”

“…The in vitro assay for Wg (19) using soluble Wg protein and the Drosophila imaginal disc cell line, clone 8 (20), has greatly contributed to biochemical analysis of the Wg signaling pathway (21)(22)(23)(24). In particular, this system led to the identification of Drosophila frizzled 2 (Dfz2) as a receptor for Wg (24).…”

Axin Contains Three Separable Domains That Confer Intramolecular, Homodimeric, and Heterodimeric Interactions Involved in Distinct Functions

“…The Dvl proteins contain a DIX domain at the N terminus and two additional highly conserved domains: a central PDZ domain, and a DEP domain (8,9). Overexpression of Dvl in cells induces the accumulation of ␤-catenin and the activation of TCF (10,11). In response to Wnt signals, Dvl forms a heterodimer with Axin through their DIX domains, leading to enhanced association of glycogen synthase kinase-3␤ with Frat and decreased phosphorylation of ␤-catenin (6,12,13).…”